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Development and Validation of a Real-Time PCR Assay for Rapid Detection of Two-Spotted Spider Mite, Tetranychus urticae (Acari: Tetranychidae).

Li D, Fan QH, Waite DW, Gunawardana D, George S, Kumarasinghe L - PLoS ONE (2015)

Bottom Line: The developed assay is rapid, detects all life stages of T. urticae within three hours, and does not react with closely related species.The assay was validated against a range of T. urticae specimens from various countries and hosts in a blind panel test.Therefore the application of the assay at New Zealand will reduce the unnecessary fumigation and be beneficial to both the importers and exporters.

View Article: PubMed Central - PubMed

Affiliation: Plant Health and Environment Laboratory, Ministry for Primary Industries, Auckland, New Zealand.

ABSTRACT
Spider mites of the genus Tetranychus are difficult to identify due to their limited diagnostic characters. Many of them are morphologically similar and males are needed for species-level identification. Tetranychus urticae is a common interception and non-regulated pest at New Zealand's borders, however, most of the intercepted specimens are females and the identification was left at Tetranychus sp. Consequently, the shipments need to be fumigated. DNA sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP) protocols could be used to facilitate the accurate identification. However, in the context of border security practiced in New Zealand, insect identifications are required to be provided within four hours of receiving the samples; thus, those molecular methods are not sufficient to meet this requirement. Therefore, a real-time PCR TaqMan assay was developed for identification of T. urticae by amplification of a 142 bp Internal Transcribed Spacer (ITS) 1 sequence. The developed assay is rapid, detects all life stages of T. urticae within three hours, and does not react with closely related species. Plasmid DNA containing ITS1 sequence of T. uritcae was serially diluted and used as standards in the real-time PCR assay. The quantification cycle (Cq) value of the assay depicted a strong linear relationship with T. urticae DNA content, with a regression coefficient of 0.99 and efficiency of 98%. The detection limit was estimated to be ten copies of the T. urticae target region. The assay was validated against a range of T. urticae specimens from various countries and hosts in a blind panel test. Therefore the application of the assay at New Zealand will reduce the unnecessary fumigation and be beneficial to both the importers and exporters. It is expected that the implementation of this real-time PCR assay would have wide applications in diagnostic and research agencies worldwide.

No MeSH data available.


Related in: MedlinePlus

Alignment of the forward primer and probe regions of ITS1 sequences from Tetranychus species.The bold black letters indicated the SNPs and the dashed line indicated the indels. Asterisk (*) denotes the tested species in the real-time PCR assay. The reverse primer is not listed as there are no SNPs among the Tetranychus species compared.
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pone.0131887.g002: Alignment of the forward primer and probe regions of ITS1 sequences from Tetranychus species.The bold black letters indicated the SNPs and the dashed line indicated the indels. Asterisk (*) denotes the tested species in the real-time PCR assay. The reverse primer is not listed as there are no SNPs among the Tetranychus species compared.

Mentions: Intra-species diversity of ITS sequences among T. urticae populations is low and their ITS sequences are completely homogenous worldwide [4,22,24]. Although ITS2 is a widely used marker for Tetranychus phylogeny and species discrimination [4,16,18,19], no suitable region could be chosen for T. urticae-specific primers and probe design. In comparison to ITS2, the ITS1 region is variable among Tetranychus species, but the intra-specific level diversity for T. urticae is low (<2%), thus it could enable us to identify a conserved region for T. urticae ITS1 sequence and provided the possibility to design a real-time PCR assay for targeting T. urticae. DNA sequence comparison showed that there are SNPs and indels in ITS1 sequences which could be targeted for primer and probe design. The ITS1 region could distinguish T. urticae from the highly similar species, T. turkestani, T. kanzawai, T. parakanzawai, T. pueriricola and T. truncatus. As a result, the T. urticae-specific real-time PCR assay was designed by targeting the ITS1 region, with SNPs and indels present in non-target Tetranychus spp. sequences in the forward primer and probe (Fig 2). This real-time PCR assay successfully detected the T. urticae species and did not cross-react with other closely related species tested. It could detect T. urticae from various countries (Tables 1 and 4), thus indicating this real-time PCR assay could be able to apply to research and biosecurity agencies worldwide in detection of T. urticae.


Development and Validation of a Real-Time PCR Assay for Rapid Detection of Two-Spotted Spider Mite, Tetranychus urticae (Acari: Tetranychidae).

Li D, Fan QH, Waite DW, Gunawardana D, George S, Kumarasinghe L - PLoS ONE (2015)

Alignment of the forward primer and probe regions of ITS1 sequences from Tetranychus species.The bold black letters indicated the SNPs and the dashed line indicated the indels. Asterisk (*) denotes the tested species in the real-time PCR assay. The reverse primer is not listed as there are no SNPs among the Tetranychus species compared.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492583&req=5

pone.0131887.g002: Alignment of the forward primer and probe regions of ITS1 sequences from Tetranychus species.The bold black letters indicated the SNPs and the dashed line indicated the indels. Asterisk (*) denotes the tested species in the real-time PCR assay. The reverse primer is not listed as there are no SNPs among the Tetranychus species compared.
Mentions: Intra-species diversity of ITS sequences among T. urticae populations is low and their ITS sequences are completely homogenous worldwide [4,22,24]. Although ITS2 is a widely used marker for Tetranychus phylogeny and species discrimination [4,16,18,19], no suitable region could be chosen for T. urticae-specific primers and probe design. In comparison to ITS2, the ITS1 region is variable among Tetranychus species, but the intra-specific level diversity for T. urticae is low (<2%), thus it could enable us to identify a conserved region for T. urticae ITS1 sequence and provided the possibility to design a real-time PCR assay for targeting T. urticae. DNA sequence comparison showed that there are SNPs and indels in ITS1 sequences which could be targeted for primer and probe design. The ITS1 region could distinguish T. urticae from the highly similar species, T. turkestani, T. kanzawai, T. parakanzawai, T. pueriricola and T. truncatus. As a result, the T. urticae-specific real-time PCR assay was designed by targeting the ITS1 region, with SNPs and indels present in non-target Tetranychus spp. sequences in the forward primer and probe (Fig 2). This real-time PCR assay successfully detected the T. urticae species and did not cross-react with other closely related species tested. It could detect T. urticae from various countries (Tables 1 and 4), thus indicating this real-time PCR assay could be able to apply to research and biosecurity agencies worldwide in detection of T. urticae.

Bottom Line: The developed assay is rapid, detects all life stages of T. urticae within three hours, and does not react with closely related species.The assay was validated against a range of T. urticae specimens from various countries and hosts in a blind panel test.Therefore the application of the assay at New Zealand will reduce the unnecessary fumigation and be beneficial to both the importers and exporters.

View Article: PubMed Central - PubMed

Affiliation: Plant Health and Environment Laboratory, Ministry for Primary Industries, Auckland, New Zealand.

ABSTRACT
Spider mites of the genus Tetranychus are difficult to identify due to their limited diagnostic characters. Many of them are morphologically similar and males are needed for species-level identification. Tetranychus urticae is a common interception and non-regulated pest at New Zealand's borders, however, most of the intercepted specimens are females and the identification was left at Tetranychus sp. Consequently, the shipments need to be fumigated. DNA sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP) protocols could be used to facilitate the accurate identification. However, in the context of border security practiced in New Zealand, insect identifications are required to be provided within four hours of receiving the samples; thus, those molecular methods are not sufficient to meet this requirement. Therefore, a real-time PCR TaqMan assay was developed for identification of T. urticae by amplification of a 142 bp Internal Transcribed Spacer (ITS) 1 sequence. The developed assay is rapid, detects all life stages of T. urticae within three hours, and does not react with closely related species. Plasmid DNA containing ITS1 sequence of T. uritcae was serially diluted and used as standards in the real-time PCR assay. The quantification cycle (Cq) value of the assay depicted a strong linear relationship with T. urticae DNA content, with a regression coefficient of 0.99 and efficiency of 98%. The detection limit was estimated to be ten copies of the T. urticae target region. The assay was validated against a range of T. urticae specimens from various countries and hosts in a blind panel test. Therefore the application of the assay at New Zealand will reduce the unnecessary fumigation and be beneficial to both the importers and exporters. It is expected that the implementation of this real-time PCR assay would have wide applications in diagnostic and research agencies worldwide.

No MeSH data available.


Related in: MedlinePlus